The overall objective of this project is to add to our current knowledge of the means by which hormonal and/or other extracellular stimuli can lead to an acceleration (or deceleration) of a specific intracellular event. Basic to this objective is a detailed understanding of the physical, kinetic, and regulatory properties of the cyclicAMP-stimulated protein kinases. The initial work will deal principally with rat and bovine brain protein kinase and its soluble-particulate distribution, specificity, and stimulation by cyclic nucleotides. The general procedure will be to isolate the various subcellular forms of the protein kinase of cerebral cortical tissue, and to characterize these forms with regard to latency, ability to be interconverted by detergents, metabolites, etc., and their kinetic properties using defined protein substrates. The specificity of these protein kinase forms with such protein substrates will be determined, and the influence of effector molecules themselves capable of binding to either the kinase or its macromolecular substrate will be evaluated. Finally, a variety of physical and catalytic probes will be utilized to characterize the mechanism by which cyclicAMP stimulates the kinase. These approaches will include isotope exchange at equilibrium measurements of the effect of the regulatory subunit on the catalytic properties of the kinase, initial rate measurements and correlation of specific activity measurements with the molecular weight changes of the kinase, fluorescence measurements and kinetic analysis of rapid molecular weight changes attending the interaction of the protein kinase-regulatory protein complex with cyclicAMP, protein substrates, and other effector molecules.